The zebrafish mutant, narrowminded (nrd), is a mutation in the gene encoding the Prdm1a (or Blimp1) transcription factor which results in a reduction in neural crest cells and downregulation of several genes required for neural crest specification at the neural plate border. The goal of this project is to determine the molecular mechanisms by which prdm1a regulates neural crest specification and neural crest specifier genes. To do this, I will determine the downstream targets of the Prdm1a transcription factor. I will use ChIP-seq to determine direct transcriptional targets of Prdm1a during neural crest specification. I will also use microarray to compare gene expression in wildtype and nrd embryos during neural crest specification. Using these techniques together, I will be able to determine and confirm novel direct targets of Prdm1a, see if they are up or down-regulated in the mutant to suggest the manner of Prdm1a regulation, and identify functional indirect targets of Prdm1a. I am also using double fluorescent RNA in situ hybridization to characterize the expression of several known neural crest specification genes that are downregulated in the nrd mutant along with prdm1a to determine if they are expressed in the same cells at the right time to be candidate direct targets. I also will use mRNA injections of candidate downstream target genes into nrd mutant embryos to attempt to rescue the mutant phenotype and place these genes within a prdm1a gene regulatory network and ChIP to determine if they are directly targeted by Prdm1a. I am additionally interested in determining the function of the PR/SET domain and Pro/Ser rich region of the Prdm1a protein and how they affect the role of Prdm1a in neural crest specification. To investigate this, I will create domain deletion constructs removing these regions and inject them into nrd embryos to see if they are able to rescue the phenotype to the same levels as wildtype prdm1a does. I will also investigate the effect of these domain deletions on expression of Prdm1a target genes using in situ hybridization and qPCR. Using these experiments, I will be able to determine the mechanism by which prdm1a regulates neural crest specification through gene regulation. PUBLIC HEALTH RELEVANCE: Neural crest defects are implicated in multiple human syndromes resulting in cleft lip and palate in approximately 6800 live births in the United States alone (March of Dimes, 2010). Understanding the genes involved in neural crest specification during embryological development is critical to the understanding of both normal and defective craniofacial development, as well as for expanding potential therapies and treatments for craniofacial disorders.